Stressful stimuli have long been known to induce reactivation of HSV-1 from latency in vivo and in vitro, yet the identity of the cellular factors responsible for the activation of viral gene expression is not known. In a related vein, the HSV-1 immediate-early regulatory protein, ICP0, is a broad and strong transcriptional activator of viral and cellular gene expression. ICP0 is required for efficient viral replication, especially at low multiplicities of infection, and for efficient reactivation from neuronal latency in an ex vivo mouse model. We have shown previously that stress-induced cellular factors can compensate for the activities of ICP0 in ICP0 null mutant-infected cells, suggesting that one function of ICP0 may be to "turn on" cellular factors induced by stress. These cellular factors include, but are likely not limited to, cyclin-dependent kinases. In Aim 1 of this project, microarray and Western blot analyses will be used to identify the cellular factors whose expression is affected by both stress and ICP0. In Aim 2, specific cellular factors affected by both stress and ICP0 will be tested for their ability to substitute for the functions of ICP0 by inhibiting their activities using siRNA and available knockout cell lines. ICP0 activates expression of all classes of HSV-1 genes. In Aim 3, the classes of viral genes activated by the stress-induced cellular, ICP0-complementing activities will be identified in cells transfected with promoter-specific reporter plasmids and subjected to stress. These experiments are designed to identify the stress-induced and ICP0-induced cellular factors that promote HSV-1 replication and may, in future experiments, assist in identifying the cellular factors responsible for reactivation of HSV-1 for neuronal latency. [unreadable] [unreadable]